Kits and methods for pathogen detection

ABSTRACT

Kits and methods for detecting pathogens without the need for laboratory equipment are disclosed. The kits and methods described herein allow for near-room temperature amplification of pathogen polynucleotides in a biological sample in a one-compartment reaction vessel. The kits and methods may be used to detect any target nucleic acid, such as DNA or RNA from a bacterial, fungal, or viral pathogen.

CROSS-REFERENCE TO RELATED APPLICATION

The benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 62/127,589, filed Mar. 3, 2015, is hereby claimed, andthe disclosure thereof is incorporated herein by reference.

FIELD OF THE INVENTION

The disclosure relates to test kits and methods for detecting pathogensin biological samples.

INCORPORATION BY REFERENCE

This application contains, as a separate part of the disclosure, asequence listing in computer-readable form (filename:49096A_SeqListing.txt, 4,395 bytes, created Mar. 1, 2016), which isincorporated by reference in its entirety.

BACKGROUND

Advances in technology relating to pathogen detection in biologicalspecimens have been instrumental in the successful management of manyinfectious diseases. Several laboratory tests for the diagnosis ofdiseases such as human papillomavirus (HPV), chlamydia and gonorrhea arecommercially available. The majority of the tests currently on themarket are based on the polymerase chain reaction (PCR), with a few thatrely on fluorescence immunoassay. These tests are extremely sensitiveand accurate, but cannot be performed outside clinical settings.Diagnostic tests based on PCR and/or immunoassays require expensivelaboratory equipment and are usually adapted for automation by large,well-equipped facilities staffed by highly-trained personnel. Thesetests are costly and may take weeks to process, creating significanthurdles for routine screening. Furthermore, the laboratoryinfrastructure and instrumentation required to run these assays are notuniversally available, limiting their dissemination. As a result,individuals without routine access to a formal healthcare system are atincreased risk of developing and dying from preventable diseases.

To increase screening rates, especially among disadvantaged populations,self-sampling techniques have been developed. For example,self-collected vaginal swabs are the currently preferred specimen typesfor chlamydia and gonorrhea testing. Self-collected samples are mailedto diagnostics laboratories for testing. Thus, it still takes weeks toobtain the results, and the reliance on expensive (and sometimes scarce)resources is not significantly reduced.

There exists a need for testing platforms and methods that are simple touse, provide rapid results, and eliminate the need for laboratoryequipment. Such cost-effective, portable tests and methods would providemeans for the detection and early treatment of pathogenic infectionsbeyond what is currently available.

SUMMARY

The disclosure is directed to test kits and methods for detectingpathogens in biological samples that do not require laboratoryinstrumentation. In one aspect, a method for detecting a pathogenpolynucleotide in a biological sample comprising an amplification stepand a detection step is provided. The amplification step comprisescombining the biological sample with (a) a padlock probe comprising a 5′end complementary to a first section of the pathogen polynucleotide anda 3′ end complementary to a second section of the pathogenpolynucleotide, wherein the first section and second section of thepathogen polynucleotide sequences are located adjacent to each other;(b) a ligase; (c) a primer comprising a polynucleotide sequencecomplementary to a portion of the padlock probe; (d) a polymerase; (e) areporter probe; and (f) a reaction buffer; to form a mixture in a singlereaction vessel. The detection step comprises wicking the mixture into atest strip and visually detecting the reporter probe on the test strip.Optionally, the amplification step does not comprise incubating themixture at a temperature greater than about 37° C. Optionally, theamplification step comprises incubating the mixture at a temperaturebetween about 20° C. and about 37° C., for example, about 30° C., forabout 20 minutes to about 2 hours.

In another aspect, the invention provides a kit for detecting a pathogenpolynucleotide in a biological sample, comprising (a) a reaction vesselcomprising: (1) a padlock probe comprising a 5′ end complementary to afirst section of the pathogen polynucleotide and a 3′ end complementaryto a second section of the pathogen polynucleotide, wherein the firstsection and second section of the pathogen polynucleotide sequence arelocated adjacent to each other; (2) a ligase; (3) a primer comprising apolynucleotide complementary to a portion of the padlock probe; (4) apolymerase; and (5) a reporter probe; (b) a reaction buffer thatsupports polynucleotide ligation and polymerization; and (c) a teststrip. In various embodiments, the reaction buffer is included in thereaction vessel.

In various aspects, the pathogen is selected from the group consistingof human papillomavirus, Chlamydia tracomatis, Neisseria gonorrhoeae,herpes simplex virus (Type 1 or Type 2), Mycoplasma genitalium,Trichomonas vaginalis, Gardnerella vaginalis, and Candida species. Inone aspect, the ligase is T4 DNA ligase. In another aspect, thepolymerase is Φ29 polymerase or Bst DNA polymerase. Combinations ofpolymerases may also be used. In still another aspect, the reactionbuffer comprises Tris-Cl, magnesium chloride, ammonium sulfate, ATP, andoptionally methanol. The reaction buffer optionally comprises about 50mM to about 1 mM Tris-Cl, about 10 mM to about 100 mM magnesiumchloride, about 5 mM to about 100 mM ammonium sulfate, about 0.1 mM toabout 10 mM ATP, and about 0% to about 20% methanol.

In various aspects, the padlock probe comprises a polynucleotidesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, and SEQ ID NO:20; the primer comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:21; and/orthe reporter probe comprises a polynucleotide sequence (a) selected fromthe group consisting of SEQ ID NO: 5, SEQ ID NO:11 and SEQ ID NO: 17 or(b) corresponding to a region of SEQ ID NO: 2, SEQ ID NO:8 or SEQ ID NO:14. All combinations of padlock probes, primers, and reporter probesdescribed herein are contemplated, as are use of probes and primerscomprising sequences having at least about 90% identity to SEQ ID NOs:2-21.

Optionally, the reporter probe is conjugated to a microparticle, forexample, a microparticle having a diameter less than about onemicrometer. In various aspects, the microparticle is a nylonmicroparticle, a gold microparticle, or a magnetic microparticle. In oneaspect, the test strip comprises filter paper, optionally filter paperhaving a pore size of about 11 micrometers. Optionally, the test stripcomprises chitosan.

In one aspect, the pathogen is HPV; the padlock probe comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:20; the primer comprisesthe polynucleotide sequence of SEQ ID NO: 6, SEQ ID NO:19, or SEQ IDNO:21; and the reporter probe comprises the polynucleotide sequence ofSEQ ID NO: 5 or a polynucleotide corresponding to a region of SEQ IDNO:2. In another aspect, the pathogen is Chlamydia tracomatis; thepadlock probe comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; theprimer comprises the polynucleotide sequence of SEQ ID NO: 12, and thereporter probe comprises the polynucleotide sequence of SEQ ID NO: 11 ora polynucleotide corresponding to a region of SEQ ID NO:8. In stillanother aspect, the pathogen is Neisseria gonorrhoeae, the padlock probecomprises a polynucleotide sequence selected from the group consistingof SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; the primer comprisesthe polynucleotide sequence of SEQ ID NO:18, and the reporter probecomprises the polynucleotide sequence of SEQ ID NO:17 or apolynucleotide corresponding to a region of SEQ ID NO:14. Probes andprimers comprising sequences having at least about 90% identity to anyof the sequences referenced herein also are contemplated.

The foregoing summary is not intended to define every aspect of theinvention, and other features and advantages of the present disclosurewill become apparent from the following detailed description, includingthe drawings. The present disclosure is intended to be related as aunified document, and it should be understood that all combinations offeatures described herein are contemplated, even if the combination offeatures are not found together in the same sentence, paragraph, orsection of this disclosure. In addition, the disclosure includes, as anadditional aspect, all embodiments of the invention narrower in scope inany way than the variations specifically mentioned above. With respectto aspects of the disclosure described or claimed with “a” or “an,” itshould be understood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. With respect toelements described as one or more within a set, it should be understoodthat all combinations within the set are contemplated. If aspects of thedisclosure are described as “comprising” a feature, embodiments also arecontemplated “consisting of” or “consisting essentially of” the feature.Additional features and variations of the disclosure will be apparent tothose skilled in the art from the entirety of this application, and allsuch features are intended as aspects of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of a method of the disclosure. First, a swabcontaining a biological sample is transferred to a reaction tubecontaining polynucleotide amplification reagents as well as reporterprobe. Second, the mixture in the reaction tube is incubated, optionallyin the hand. The padlock probe hybridizes to target pathogenpolynucleotides via a 5′ complementary end and a 3′ complementary end.Ligation occurs, forming a circular padlock probe, to which a primeranneals. Rolling circle amplification (RCA) occurs, generating antisensecopies of the padlock probe. The reporter probe comprises apolynucleotide sequence complementary to the newly generated antisensecopies of the padlock probe and hybridizes to the RCA product. Third, atest strip is inserted in the reaction tube. The mixture in the reactiontube is wicked through the test strip and a positive or negative resultis visualized.

FIG. 2A and FIG. 2B depict representative test strip results of acompleted assay. FIG. 2A depicts a negative result with no signal belowthe control band (arrow), indicating the pathogen polynucleotide is notpresent in the biological sample, and FIG. 2B depicts a positive result(asterisk) below the control band (arrow), indicating a pathogenpolynucleotide is present in the biological sample.

DETAILED DESCRIPTION

The present disclosure provides kits and methods for detecting pathogensin biological samples that eliminate the need for laboratory equipment.The kits and methods provide a solution to diagnosing infection andmanaging public health in medically underserved populations worldwide,while specifically addressing health challenges faced by developingcommunities.

The kits and methods of the disclosure provide numerous advantagescompared to currently marketed tests. The kits and methods of thedisclosure use isothermal rolling-circle amplification (RCA) in order toeliminate reverse transcription and allow for near-room temperatureamplification of polynucleotides in a one-compartment reaction vessel,allowing for on-site detection of target pathogen polynucleotideswithout requiring expensive instrumentation, technical training, orcomplex protocols (FIG. 1). Therefore, unlike other methods for pathogendetection, the kits and methods of the disclosure do not require anylaboratory equipment such as PCR machines or separation columns.

Additionally, the polynucleotide amplification reactions can be run atconstant temperatures (e.g., staying within five degrees from thestarting temperature) that are near ambient or room temperature, forexample, about 20° C. to about 37° C. The reactions can thus beperformed using, for example, body heat such as the heat from one'shand, rather than requiring temperature cycling for different steps ofthe reaction and/or heated water baths or incubators.

The reactions can be run in a single reaction vessel, using amulti-functional buffer that supports both polynucleotide ligation andpolymerization reactions, eliminating the need for sequential processsteps requiring the addition of reagents in series, or multiple separatereaction compartments. The test strip serves as a separation device thatdetects reporter probes hybridized to amplified target nucleic acidsbased on size exclusion, eliminating the need for capture probesembedded in the test strip. Because the amplification can be completedin as little as 20-30 minutes, virtually immediate results can beprovided on-site, rather than requiring days or weeks for results to bereturned from a clinician or laboratory. After the tests are completed,the reaction vessel and test strips can be easily disposed of withoutrisk of contamination because the RCA products are non-replicating,unlike amplicons produced using PCR or other methods.

The kits and methods of the disclosure thus overcome many cultural,geographic, and economic barriers that currently preclude adequatedisease screening. Use of the kits and methods of the present disclosureas a primary screening strategy improves disease prevention andtreatment. Rapid testing eliminates the need for follow-up consultationsand allows immediate treatment, when necessary. The kits and methods ofthe disclosure are useful for rapid on-site disease detection indoctor's offices, point of care facilities, community clinics,pharmacies, hospitals, ambulances and other first responder vehicles(e.g., firetrucks, helicopters, airplanes, etc.), and at home. The kitsand methods are also useful in preventing and monitoring food- andwater-borne illnesses due to contamination by pathogens.

The kits and methods of the disclosure are applicable to DNA and RNA,whether single-stranded or double-stranded. Any bacterial, fungal, orviral pathogen may be detected using the kits and methods of thedisclosure. For example, in one aspect, the kits and methods are used todetect a bacterial pathogen including, but not limited to, bacteriabelonging to the genus Bacillus (e.g., B. anthracis), Bordetella (e.g.B. pertussis), Borrelia (e.g., B. burgdorferi), Brucella (e.g., B.abortus, B. canis, B. melitensis, B. suis), Campylobacter (e.g., C.jejuni), Chlamydia (e.g., C. pneumonia, C. psittaci, C. trachomatis),Clostridium (e.g., C. botulinum, C. difficile, C. perfringens),Corynebacterium (e.g., C. diphtheria), Enterococcus (e.g., E. faecalis),Escheria (e.g., E. coli), Gardnerella (e.g., G. vaginalis), Haemophilus,(e.g., H. influenza), Helicobacter (e.g., H. pylori), Legionea (e.g., L.pneumophila), Listeria, (e.g., L. monocytogenes), Mycoplasma (e.g., M.genitalium), Neisseria (e.g., N. gonorrhoeae, N. meningitides),Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhi, S.typhimurium), Staphylococcus (e.g., S. aureus), Streptococcus (e.g, S.pneumonia), or Vibrio (e.g., V. cholerae). In another aspect, the kitsand methods are used to detect a fungal pathogen including, but notlimited to, fungi belonging to the genus Aspergillus, Blastomyces,Candida, Cladosporium, Coccidioides, Cryptococcus, Exserohilum,Histoplasma, Mucoromycotina, Pneumocystis, Sporothrix, or Stachybotrys.In still another aspect, the kits and methods are used to detect a viralpathogen including, but not limited to, adeno-associated virus, denguevirus, Ebolavirus, encephalomyocarditis virus, Epstein-Barr virus,hepatitis virus, herpesvirus (e.g., herpes simplex virus Type 1 or Type2), human immunodeficiency virus (HIV), HPV, influenza virus, MERScoronavirus, measles virus, mumps virus, Norovirus, poliovirus,rotavirus, rubella virus, West Nile virus, and yellow fever virus, or aprotozoan pathogen including, but not limited to, Trichomonas vaginalis.The genomes of the representative pathogens referenced above are knownin the art, and generation of a padlock probe with complementarysequences suitable for hybridization is well within the skill of theart. In one aspect, the pathogen polynucleotide comprises a targetpolynucleotide selected from the genome of HPV, Chlamydia tracomatis, orNeisseria gonorrhoeae, such as the polynucleotide of SEQ ID NO: 1, SEQID NO:7, or SEQ ID NO:13.

The following definitions may be useful in aiding the skilledpractitioner in understanding the disclosure. Unless otherwise definedherein, scientific and technical terms used in the present disclosureshall have the meanings that are commonly understood by those ofordinary skill in the art.

The term “capture region” refers to a region on a conventional teststrip containing immobilized capture probes which bind to a targetpolynucleotide and allow for visual detection of the targetpolynucleotide.

The term “constant temperature” refers to temperatures that are within±5° C. of a reference temperature.

The term “microparticle” refers to a particle comprising a diameter lessthan 100 micrometers and includes particles having a diameter less thanone micrometer. Microparticles may be spherical (e.g., microbeads) orhave an irregular shape, and may be composed of any of a number ofsubstances, including gold and/or other metals, nylon and/or otherpolymers, magnetic compounds, and combinations thereof.

The term “padlock probe” refers to a single-stranded polynucleotidewhose 5′ and 3′ ends are complementary to a target polynucleotidesequence, for example, as described in Nilsson et al., Science265(5181):2085-2088 (1994), incorporated herein by reference.

The term “primer” refers to a polynucleotide that hybridizes to a targetpolynucleotide sequence and serves as the starting point for synthesisof new polynucleotides.

The term “rolling circle amplification” or “RCA” refers to theisothermal amplification of a circularized probe, for example, asdescribed in U.S. Pat. No. 5,854,033, incorporated herein by reference.

In one aspect, the disclosure provides a method for detecting a pathogenpolynucleotide in a biological sample comprising an amplification stepcomprising combining the biological sample with (1) a padlock probecomprising a 5′ end complementary to a first section of the pathogenpolynucleotide and a 3′ end complementary to a second section of thepathogen polynucleotide, wherein the first section and the secondsection of the pathogen polynucleotide are adjacent to each other; (2) aligase; (3) a primer comprising a polynucleotide sequence complementaryto a portion of the padlock probe; (4) a polymerase; (5) a reporterprobe; and (6) a reaction buffer. A detection step is then performed.

In another aspect, the disclosure provides a test kit for detection of apathogen polynucleotide in a biological sample comprising a reactionvessel comprising: (1) a padlock probe comprising a 5′ end complementaryto a first section of the pathogen polynucleotide sequence and a 3′ endcomplementary to a second section of the pathogen polynucleotidesequence, wherein the first section and second section of pathogenpolynucleotide sequences are located adjacent to each other; (2) aligase that anneals the 5′ and 3′ ends of the padlock probe to form acircular padlock probe; (3) a primer comprising a polynucleotidesequence complementary to a portion of the padlock probe; (4) apolymerase; and (5) a reporter probe. The kit further comprises areaction buffer that supports polynucleotide ligation and polymerizationand a test strip for detecting the reporter probe.

The biological sample is, in various embodiments, obtained from a humanor other mammalian subject, for example, by collecting a bodily fluidsample or swabbing a body orifice. The sample may be collected by, e.g.,a health care work or self-sampling. Alternatively, the biologicalsample is obtained from an environmental source, such as a water orsoil. The biological sample may also be a food sample (e.g., a fluid orswab taken from food in order to, for example, detect contamination).The biological sample and reagents (padlock probe, ligase, primer,polymerase, reporter probe, and reaction buffer) are combined to form amixture in a single reaction vessel, such as a test tube.

When the mixture of the biological sample and reagents is formed, if thetarget pathogen polynucleotide is present in the biological sample, the5′ end and 3′ end of the padlock probe hybridize to adjacent first andsecond sections of the pathogen polynucleotide sequence. In variousaspects, the padlock probe comprises a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:20. In one aspect, the padlock probe comprises a 5′section comprising the sequence in SEQ ID NO:3, SEQ ID NO:9, or SEQ IDNO:15 and/or a 3′ section comprising the sequence set forth in SEQ IDNO:4, SEQ ID NO:10, or SEQ ID NO:16. In another aspect, the padlockprobe comprises the polynucleotide sequences of SEQ ID NO:3 and SEQ IDNO:4, SEQ ID NO:9 and SEQ ID NO:10, or SEQ ID NO:15 and SEQ ID NO:16. Instill another aspect, the padlock probe comprises the polynucleotidesequence of SEQ ID NO:2, SEQ ID NO: 8, SEQ ID NO:14, or SEQ ID NO:20.Variants comprising a nucleic acid sequence comprising at least about80%, at least about 90%, or at least about 95% sequence identity to thesequences referenced above also may be used in the context of theinvention.

The hybridization of the padlock probe to the pathogen polynucleotidebrings the 5′ end and 3′ end of the padlock probe in close proximity,allowing the ligase to join the 5′ end and 3′ end of the padlock probetogether to form a circular padlock probe. It will be appreciated thatthe sequences above are merely examples of sequences suitable for use ina padlock probe, and other sequences are suitable for use so long as thesequences hybridize to the target polynucleotide in such a manner toallow a ligase to generate a circular padlock probe. In one aspect, theligase is an enzyme that can ligate polynucleotide strands at atemperature at or below 37° C., for example T4 DNA ligase. The targetpathogen polynucleotide is only briefly required to serve as a bridge tocircularize the padlock probe for subsequent amplification.

Once the ligase has circularized the padlock probe, the primer, whichcomprises a polynucleotide sequence complementary to a portion of thepadlock probe, hybridizes to the circular padlock probe to initiatereplication of a single-stranded polynucleotide sequence containingrepetitive, antisense copies of the padlock probe. In various aspects,the primer comprises a polynucleotide sequence set forth in SEQ ID NO:6,SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21, or apolynucleotide sequence complementary to a portion of SEQ ID NO:2, SEQID NO:8, SEQ ID NO:14 or SEQ ID:20. Primers comprising a sequence atleast 90% identical to these sequences also may be used in variousembodiments. The replication of single-stranded antisense copies of thepadlock probe is mediated by the polymerase. In one aspect, thepolymerase is capable of working on small circular polynucleotides, forexample, a polymerase derived from a bacteriophage or bacterium, such asphi29 (Φ29) polymerase or Bacillus stearothermophilus (Bst) DNApolymerase (e.g., Bst DNA polymerase, large fragment).

The reporter probe hybridizes to the antisense copies of the padlockprobe to form a reporter complex. In one aspect, the reporter probecomprises a polynucleotide sequence identical to a region of the padlockprobe, such as the region adjacent to the 5′ end of the padlock probethat is complementary to the pathogen polynucleotide. Optionally, thereporter probe comprises a polynucleotide sequence identical to a regionof at least 10 polynucleotides of the padlock probe, for example, atleast 10 polynucleotides, at least 15 polynucleotides, at least 20polynucleotides, at least 25 polynucleotides, or at least 30polynucleotides. In various aspects, the reporter probe comprises apolynucleotide corresponding to a portion of SEQ ID NO:2, SEQ ID NO:8,SEQ ID NO:14, or SEQ ID NO:20, for example, in some embodiments, thereporter probe comprises the polynucleotide sequence of SEQ ID NO:5, SEQID NO:11, or SEQ ID NO:17. Other suitable reporter probes may begenerated using routine laboratory methods.

Optionally, the reporter probe is conjugated to a microparticle. In oneaspect, the microparticle has a diameter less than about one micrometer.In various aspects, the microparticle is selected from a nylonmicroparticle, gold microparticle, or magnetic (e.g., paramagnetic)microparticle. Combinations of microparticles may also be used.Conjugation of the reporter probe and microparticle can be achievedusing any suitable method, such as covalent linkage.

The reaction buffer of the disclosure is capable of supporting both thepolynucleotide ligation and polymerization reactions, therebyeliminating the need for more than one buffer, a washing step, ormultiple reaction compartments. In one aspect, the reaction buffercomprises Tris-Cl, magnesium chloride, ammonium sulfate, ATP, andoptionally methanol, optionally about 10 mM to about 100 mM Tris-Cl,about 2 mM to about 10 mM magnesium chloride, about 1 mM to about 10 mMammonium sulfate, about 0.1 mM to about 1 mM ATP, pH of about 7.0 toabout 7.6, optionally in about 20% v/v methanol. For example, in oneaspect, a 10× reaction buffer comprises about 500 mM to about 1 MTris-HCl, about 100 mM magnesium chloride, and about 50 mM to about 100mM ammonium sulfate, pH about 7.5 at 25° C., optionally furthercomprising about 40 mM DTT and/or about 1 mM ATP. In one aspect, theamplification step is performed at a temperature less than about 37° C.,unlike traditional PCR reactions, which require laboratory equipment toachieve temperatures greater than 90° C. Therefore, in one aspect, theamplification step does not comprise incubating the mixture at atemperature greater than about 37° C. In one aspect, the amplificationstep comprises incubating the mixture at a temperature between about 20°C. and about 37° C., for example, between about 22° C. and about 35° C.,between about 23° C. and about 32° C., or between about 25° C. and about30° C. In another aspect, the amplification step does not compriseincubating the mixture at a temperature greater than about 30° C.Optionally, the amplification step comprises incubating the mixture at aconstant temperature of about 30° C. The amplification reaction can,therefore, be performed using only body heat, for example, by holdingthe reaction vessel in the palm of one's hand. In one aspect, theamplification step comprises incubating the mixture at a temperatureless than 37° C. for a time of about 20 minutes to about 2 hours, forexample, about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 60 minutes, about 90 minutes or about 2 hours.

In one aspect, a method of the disclosure further comprises a detectionstep comprising wicking the mixture, e.g., via capillary action, into atest strip and visually detecting the reporter probe. In one aspect ofthe kits and methods described herein, the test strip is a paper strip,optionally comprising filter paper, such as Whatman #1 filter paper. Thetest strip optionally comprises pores having a diameter of about 5micrometers to about 20 micrometers, for example, about 5 micrometers,about 10 micrometers, about 11 micrometers, about 12 micrometers, about13 micrometers, about 14 micrometers, about 15 micrometers, or about 20micrometers. Optionally, the test strip comprises a region comprisingchitosan, which non-specifically binds polynucleotides and provides acontrol region or indicator of test completion. The test strip separatesthe components in the mixture based on size exclusion so that reporterprobes hybridized to amplified polynucleotides, i.e., the reportercomplexes, travel less along the length of the test strip than smaller,uncomplexed reporter probes. Thus, when the target pathogenpolynucleotide is present in the biological sample, resulting in theproduction of antisense copies of the padlock probe that hybridize tothe reporter probe, a distinct band is visible near the bottom of thetest strip, e.g., below an indicator of test completion (FIG. 2B),indicating pathogen polynucleotide is present in the biological sample.In contrast, a test strip dipped into a mixture containing onlyuncomplexed reporter probes exhibits a band farther up the test strip,e.g., at the mid-point of the strip or at an indicator of testcompletion, indicating the target pathogen polynucleotide is not presentin the biological sample (FIG. 2A). Because the test strip separates themixture based on size exclusion, no capture region or capture probesembedded in the test strip are required.

In one aspect of the kits and methods of the disclosure, the pathogen isHPV; the padlock probe comprises a polynucleotide sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:20; the primer comprises the polynucleotide sequence of SEQ IDNO:6, SEQ ID NO:19, or SEQ ID NO:21; and the reporter probe comprisesthe polynucleotide sequence of SEQ ID NO:5 or a polynucleotidecorresponding to a region of SEQ ID NO:2 or SEQ ID NO:20. In anotheraspect, the pathogen is Chlamydia tracomatis, the padlock probecomprises a polynucleotide sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; the primer comprises thepolynucleotide sequence in SEQ ID NO:18; and the reporter probecomprises the polynucleotide sequence set forth in SEQ ID NO:11 or apolynucleotide corresponding to a region of SEQ ID NO:8.

In another aspect, the pathogen is Chlamydia tracomatis and the padlockprobe hybridizes to a target region inside the cryptic plasmid DNA, forexample, the 122-basepair or 140-basepair region within the crypticplasmid targeted by the REALTIME CT/NG assay (Abbott Molecular, DesPlaines, Ill.), as described in Centers for Disease Control andPrevention, “Recommendations for the Laboratory-Based Detection ofChlamydia trachomatis and Neisseria gonorrhoeae.” Recommendations andReports 63, No. 2 (2014) and Gaydos et al., J. Clin. Microbiol. 48(9):3236-3243 (2010), incorporated herein by reference. In one aspect, twopadlock probes, for example, to target both the 122-bp and 140-bpregions in the cryptic plasmid, are used.

In still another aspect, the pathogen is Neisseria gonorrhoeae, thepadlock probe comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, theprimer comprises the polynucleotide sequence set forth in SEQ ID NO:18,and the reporter probe comprises the polynucleotide sequence set forthin SEQ ID NO:17 or a polynucleotide corresponding to a region of SEQ IDNO: 14. In one aspect, the pathogen is Neisseria gonorrhoeae, and thepadlock probe hybridizes to a target region within the Opa gene or theM.NgoPII gene, such as a target region described in “Recommendations forthe Laboratory-Based Detection of Chlamydia trachomatis and Neisseriagonorrhoeae,” supra.

In another aspect, the pathogen is herpes simplex virus Type 1 or Type2, and the padlock probe hybridizes to a target region within theglycoprotein B gene, for example, a target region described in Kim etal. J. Clin. Virol. 50: 26-30 (2012); Van Der Pol et al., J. Clin.Microbiol 50: 3466-3471 (2012); and Emmadi et al., J Mol Diagn 13:583-604 (2011), incorporated herein by reference.

In one aspect, the pathogen is Mycoplasma genitalium, and the padlockprobe hybridizes to a target region within the MG190 gene or the pdhDgene, for example, a target region described in McGowin et al., Infect.Immun. 80: 3842-3849 (2012) or Müller et al., J. Clin. Microbiol.88:311-315 (2012), incorporated herein by reference.

In another aspect, the pathogen is Trichomonas vaginalis or Gardnerellavaginalis, and the padlock probe hybridizes to a target region withinthe 16S rRNA gene, for example, a target region described in Andrea etal., J. Clin. Microbiol. 49(3): 866-869 (2011); Cartwright et al., J.Clin. Biol. 50: 2321-2329 (2012); or Fredricks et al., J. Clin.Microbiol. 45: 3270-3276 (2007), incorporated herein by reference.

In still another aspect, the pathogen is a Candida species (e.g., C.albicans or C glabrata), and the padlock probe hybridizes to a targetregion on the internal transcribed spacer 2 (ITS2), for example, atarget region described in Cartwright et al., J. Clin. Biol. 51:3694-3699 (2013), incorporated herein by reference.

In various aspects, the kits and methods of the disclosure comprise avariant of a polynucleotide described herein, the variant having anucleic acid sequence comprising at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to any of thepolynucleotides set forth in SEQ ID NOs: 1-21.

The following example is provided by way of illustration and is notintended to be limiting.

Example

Design of HPV Probe Set for RCA Product Detection.

A padlock probe was designed to include 5′ and 3′ target recognitionsequences that, when hybridized to target HPV E6 mRNA, would bring the5′ and 3′ ends of the padlock probe together for subsequent ligation.The extended sequence between the target-homologous regions of the RCAproduct provided a convenient hybridization site for the reporterprobes. The reporter probe served as the positive control byaccumulating at a control site downstream of the detection zone that wasloaded with chitosan for non-specific capture of excess reporter probe.The reporter probe design included a primary amine at the 3′ terminusfor conjugation to N-hydroxysuccinimide (NHS)-functionalized reporter.Table 1 shows the synthetic oligonucleotides used for the HPV assay.

TABLE 1 HPV E6 RNA 84-TGCACCAAAAGAGAACTGCAATGT-107 (SEQ ID NO: 1)target region analog Padlock for Full sequence: HPVpTCTTTTGGTGCATTTATTTCCTCAATGCTGCTGCTGTACTACTAGTGATTTACTTGGATGTCTACATTGCAGTTC (SEQ ID NO: 2) 5′end antisense to target: TCTTTTGGTGCA (SEQ ID NO: 3) 3′end antisense to target: ACATTGCAGTTC (SEQ ID NO: 4) Reporter probeTTTATTTCCTCAATGCTGCTGCTGTA (SEQ ID NO: 5) PrimerAGCAGCAGCATTGAGGAAAT (SEQ ID NO: 6) All sequences are written 5′-3′.Additional oligonucleotides suitable for an HPV assay include (written5′-3′) the primer TGCTGCCGGTCACTTAACAT (SEQ ID NO: 19), the padlockpCATCTGAAAAAATTTTTATGTTAAGTGACCGGCAGCATTTTTTCTAATCTGAAGCTTGGTGGACTCTTTTTTTCACTAGGCAGCC (SEQ ID NO: 20), and the HPV type 16 gapprimer pAAAGAGA (SEQ ID NO: 21).

Reporter Conjugation Strategy.

For optimal visualization and contrast on the white paper platform,paramagnetic microbeads were chosen as the visual reporter. Bypurchasing microbeads that were pre-functionalized withN-hydroxysuccinimide (NHS), the reporter probes and microbeads werecovalently linked without additional reagents. Unreacted probe wasrecovered via magnetic isolation of the nanoparticles. Conjugation ofthe microbeads was performed according to the manufacturer's protocol.Briefly, 300 pL of 10 mg/mL of magnetic microbeads (1 pm average; ThermoFisher Pierce, Rockford, Ill.) was transferred to a 1.5 mL Eppendorftube and magnetically isolated. Following removal of the storage medium,the beads were resuspended in 1 mL of 1 mM HCl in Milli-Q water andvortexed gently for 15 seconds. Following magnetic isolation, thesupernatant was removed prior to adding approximately 100 nmoles ofaminated reporter probe suspended in a 300 pL aliquot of 50 mM borate,pH 8.5. The reaction was allowed to proceed for 2 hours to 3 hours atroom temperature prior to magnetic isolation of the beads and removal ofthe supernatant. The beads were then resuspended in 1 mL of 100 mMglycine pH 2.0 and gently vortexed for 15 seconds. This step wasrepeated 3 times prior to rinsing the beads with Milli-Q water using thesame methodology. Beads were then stored in Milli-Q water for subsequentuse.

RCA of Synthetic HPV E6 mRNA.

The typically separate ligation and polymerization steps were combinedinto a single reaction. The 10× Φ29 polymerase buffer was eliminated,and instead, 5 pL of 10× T4 ligase buffer (1 M Tris-Cl, 100 mM magnesiumchloride, 50 mM ammonium sulfate, 10 mM ATP, pH 7.4) was combined with400 units of T4 ligase, 160 pM padlock probe, 80 pM synthetic HPV targetmRNA, 200 pM each dNTPs, 240 pM primer, 20 pg bovine serum albumin, 10units of 129 DNA polymerase, and 50 pg of reporter probe-functionalizedmicrobeads to a total volume of 50 pL using sterile Milli-Q watercontaining 0.1% (v/v) diethyl pyrocarbonate (DEPC). The final 1×reaction buffer comprised 100 mM Tris-Cl, 10 mM magnesium chloride, 5 mMammonium sulfate, 1 mM ATP, pH 7.4, in 20% v/v methanol. The RCAreaction was then allowed to proceed for one hour at 30° C. Large-scaleamplification of synthetic HPV E6 mRNA from concentrations as low as 600pM and detectable amplification after only 30 minutes were alsoachieved.

Detection of RCA Product on Paper Strips.

For visual indication of product, magnetic microbeads were chosen due totheir intrinsically dark-brown coloration. The microbeads wereconjugated to a reporter probe antisense to the RCA product in order toprovide high-contrast resolution against the white background of theWhatman #1 filter paper strips. Due to the large size of the RCAproduct, the 11 micron particle retention of the Whatman #1 filter paperretarded the product fairly quickly after initial wicking. Themicrobead/product complex formed a distinct band approximately 5 mm fromthe bottom of the paper strip due simply to size exclusion of the largeproduct DNA strand within the porous paper (FIG. 2A). A positive assaycontrol band was also included and consisted of a narrow band ofimmobilized chitosan applied 10 mm above the bottom of the strip. Byincluding the functionalized microbeads in the RCA mixture,hybridization occurred during the amplification phase for immediateproduct visualization upon introduction of the paper strip. The upperchitosan band captured remaining functionalized microbeads due tonon-specific adsorption of DNA to indicate that the test workedappropriately.

Chlamydia trachomatis. The design strategy and protocol described abovefor HPV was also used to develop an assay for detecting polynucleotidesfrom the pathogen Chlamydia trachomatis. Table 2 shows the syntheticoligonucleotides used for the Chlamydia assay.

TABLE 2 C. trachomatis 16S 79-ACGATTGTTTAGTGGCGGA-98 (SEQ ID NO: 7)rRNA target region analog Padlock Probe Full sequence: for ChlamydiapAACAATCGTTTTATTTCCTCAATGCTGCTGCTGTACTACTAGTGATTTACTTGGGATGTCTTCCGCCACTA (SEQ ID NO: 8) 5′region antisense to target: AACAATCGT (SEQ ID NO: 9) 3′region antisense to target: TCCGCCACTA (SEQ ID NO: 10) Reporter probeTTTATTTCCTCAATGCTGCTGCTGTA (SEQ ID NO: 11) PrimerAGCAGCAGCATTGAGGAAAT (SEQ ID NO: 12) All sequences are written 5′-3′.

Neisseria gonorrhoeae assay. The design strategy and protocol describedabove for HPV was used to develop an assay for detecting gonorrhea.Table 3 shows the synthetic oligonucleotides used for the gonorrheaassay.

TABLE 3 N. gonorrhoeae 16S 560-ACTGCGTTCTGAACTGGGTG-774 (SEQ ID NO: 13)RNA target region analog Padlock Probe Full sequence: for N.pAGAACGCAGTTTTATTTCCTCAATGCTGCTGCTGTACTACTAG gonorrhoeaeTGATTTACT TGGATGTCTCACCCAGTTC (SEQ ID NO: 14) 5′end antisense to target: AGAACGCAGT (SEQ ID NO: 15) 3′end antisense to target: CACCCAGTTC (SEQ ID NO: 16) Reporter probeTTTATTTCCTCAATGCTGCTGCTGTA (SEQ ID NO: 17) PrimerAGCAGCAGCATTGAGGAAAT (SEQ ID NO: 18) All sequences are written 5′-3′.

The foregoing Example demonstrates that the kits and methods describedherein allow for rapid and portable detection of a pathogen in abiological sample without the use of laboratory instrumentation. Thedesign strategy and protocol provided above is applicable to otherpathogens of interest including, but not limited to, the pathogensdescribed herein.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this disclosure that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed:
 1. A kit for detection of a pathogen polynucleotide ina biological sample comprising: (a) a reaction vessel comprising: (1) apadlock probe comprising a 5′ end complementary to a first section ofthe pathogen polynucleotide and a 3′ end complementary to a secondsection of the pathogen polynucleotide, wherein the first section andsecond section of the pathogen polynucleotide sequence are locatedadjacent to each other; (2) a ligase that anneals the 5′ and 3′ ends ofthe padlock probe together to form a circular padlock probe; (3) aprimer comprising a polynucleotide complementary to a portion of thepadlock probe; (4) a polymerase; and (5) a reporter probe; (b) areaction buffer that supports polynucleotide ligation andpolymerization; and (c) a test strip.
 2. The kit of claim 1, wherein thepathogen is selected from the group consisting of human papillomavirus,Chlamydia tracomatis, and Neisseria gonorrhoeae.
 3. The kit of claim 1or 2, wherein the padlock probe comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:20, and a variant having at least 90%sequence identity to any of the foregoing.
 4. The kit of any of claims1-3, wherein the padlock probe comprises the polynucleotide sequences ofSEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:15and SEQ ID NO:16, or variants having at least 90% sequence identity toany of the foregoing pairs.
 5. The kit of any of claims 1-4, wherein thepadlock probe comprises the polynucleotide sequence of SEQ ID NO:2, SEQID NO: 8, SEQ ID NO:14, SEQ ID NO:20, or a variant having at least 90%sequence identity to any of the foregoing.
 6. The kit of any of claims1-5, wherein the ligase is T4 DNA ligase.
 7. The kit of any of claims1-6, wherein the primer comprises the polynucleotide sequence of SEQ IDNO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, or avariant having at least 90% sequence identity to any of the foregoing.8. The kit of any of claims 1-7, wherein the polymerase is 129polymerase or Bst DNA polymerase.
 9. The kit of any of claims 1-8,wherein the reporter probe comprises a polynucleotide sequence (a)selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ IDNO:17, and a variant having at least 90% sequence identity to any of theforegoing or (b) corresponding to a region of SEQ ID NO:2, SEQ ID NO:8,SEQ ID NO:14, or SEQ ID NO:20.
 10. The kit of any of claims 1-9, whereinthe reporter probe comprises a polynucleotide sequence identical to thepolynucleotide sequence of the padlock probe adjacent to the 5′complementary end.
 11. The kit of any of claims 1-10, wherein thereporter probe is conjugated to a microparticle.
 12. The kit of claim11, wherein the microparticle has a diameter less than about onemicrometer.
 13. The kit of claim 11 or 12, wherein the microparticle isa nylon microparticle, a gold microparticle, or a magneticmicroparticle.
 14. The kit of any of claims 1-13, wherein the reactionbuffer comprises Tris-Cl, magnesium chloride, ammonium sulfate, ATP, andmethanol.
 15. The kit of any of claims 1-14, wherein the reaction buffercomprises about 100 mM Tris-Cl, about 10 mM magnesium chloride, about 5mM ammonium sulfate, about 1 mM ATP, pH about 7.4, optionally in about20% v/v methanol.
 16. The kit of any of claims 1-14, wherein thereaction buffer comprises about 50 mM Tris-Cl, about 10 mM magnesiumchloride, about 10 mM ammonium sulfate, about 4 mM DTT, pH about 7.5,optionally further comprising about 0.1 mM to about 1 mM ATP.
 17. Thekit of any of claims 1-16, wherein the test strip comprises filterpaper, optionally filter paper comprising a pore size of about 11micrometers.
 18. The kit of any of claims 1-17, wherein the test stripcomprises chitosan.
 19. The kit of any of claims 1-18, wherein thepathogen is human papillomavirus; the padlock probe comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, and a variant having atleast 90% sequence identity to any of the foregoing; the primercomprises the polynucleotide sequence of SEQ ID NO: 6, SEQ ID NO:19, SEQID NO:21, or a variant having at least 90% sequence identity to SEQ IDNO:6, SEQ ID NO:19, or SEQ ID NO:21; and the reporter probe comprisesthe polynucleotide sequence of SEQ ID NO: 5 or a variant having at least90% sequence identity to SEQ ID NO:5, or a polynucleotide correspondingto a region of SEQ ID NO:2 or SEQ ID NO:20.
 20. The kit of any of claims1-18, wherein the pathogen is Chlamydia tracomatis; the padlock probecomprises a polynucleotide sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and a variant having at least90% sequence identity to any of the foregoing; the primer comprises thepolynucleotide sequence of SEQ ID NO: 12 or a variant having at least90% sequence identity to SEQ ID NO:12, and the reporter probe comprisesthe polynucleotide sequence of SEQ ID NO:11 or a variant having at least90% sequence identity to SEQ ID NO:11, or a polynucleotide correspondingto a region of SEQ ID NO:8.
 21. The kit of any of claims 1-18, whereinthe pathogen is Neisseria gonorrhoeae; the padlock probe comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and a variant having at least 90%sequence identity to any of the foregoing; the primer comprises thepolynucleotide sequence of SEQ ID NO:18 or a variant having at least 90%sequence identity to SEQ ID NO:18, and the reporter probe comprises thepolynucleotide sequence of SEQ ID NO:17 or a variant having at least 90%sequence identity to SEQ ID NO:17, or a polynucleotide corresponding toa region of SEQ ID NO:14.
 22. A method for detecting a pathogenpolynucleotide in a biological sample comprising: (1) an amplificationstep comprising (i) combining the biological sample with (a) a padlockprobe comprising a 5′ end complementary to a first section of thepathogen polynucleotide and a 3′ end complementary to a second sectionof the pathogen polynucleotide, wherein the first section and secondsection of the pathogen polynucleotide sequences are located adjacent toeach other; (b) a ligase; (c) a primer comprising a polynucleotidesequence complementary to a portion of the padlock probe; (d) apolymerase; (e) a reporter probe; and (f) a reaction buffer; to form amixture in a single reaction vessel, and (ii) incubating the mixture fora period of time; and (2) a detection step comprising wicking themixture into a test strip and visually detecting the reporter probe onthe test strip.
 23. The method of claim 22, wherein the amplificationstep does not comprise incubating the mixture at a temperature greaterthan about 37° C.
 24. The method of claim 22 or 23, wherein theamplification step comprises incubating the mixture at a temperaturebetween about 20° C. and about 37° C.
 25. The method of any of claims22-24, wherein the amplification step does not comprise incubating themixture at a temperature greater than about 30° C.
 26. The method of anyof claims 22-25, wherein the amplification step comprises incubating themixture at a constant temperature of about 30° C.
 27. The method of anyof claims 22-26, wherein the amplification step comprises incubating themixture for about 20 minutes to about 2 hours.
 28. The method of any ofclaims 22-27, wherein the amplification step and/or the detection stepis performed without additional instrumentation.
 29. The method of anyof claims 22-28, wherein the pathogen is human papillomavirus, Chlamydiatracomatis, or Neisseria gonorrhoeae.
 30. The method of any of claims22-29, wherein the pathogen polynucleotide comprises the polynucleotidesequence of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, or a variant havingat least 90% sequence identity to any of the foregoing.
 31. The methodof any of claims 22-30, wherein the padlock probe comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, and a varianthaving at least 90% sequence identity to any of the foregoing.
 32. Themethod of any of claims 22-31, wherein the padlock probe comprises thepolynucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:9 andSEQ ID NO:10, SEQ ID NO:15 and SEQ ID NO:16, or variants having at least90% sequence identity to any of the foregoing pairs.
 33. The method ofany of claims 22-32, wherein the padlock probe comprises thepolynucleotide sequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQID NO:20, or a variant having at least 90% sequence identity to any ofthe foregoing.
 34. The method of any of claims 22-33, wherein the ligaseis T4 DNA ligase.
 35. The method of any of claims 22-34, wherein theprimer comprises a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQID NO:21, and a variant having at least 90% sequence identity to any ofthe foregoing.
 36. The method of any of claims 22-35, wherein thepolymerase is 129 polymerase or Bst DNA polymerase.
 37. The method ofany of claims 22-36, wherein the reporter probe comprises apolynucleotide sequence (a) selected from the group consisting of SEQ IDNO:5, SEQ ID NO:11 SEQ ID NO:17, and a variant having at least 90%sequence identity to any of the foregoing, or (b) corresponding to aregion of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, or avariant having at least 90% sequence identity to any of the foregoing.38. The method of any of claims 22-37, wherein the reporter probecomprises a polynucleotide sequence identical to the region of thepadlock probe adjacent to the 5′ complementary end.
 39. The method ofany of claims 22-38, wherein the reporter probe is conjugated to amicroparticle.
 40. The method of claim 39, wherein the microparticle hasa diameter less than about one micrometer.
 41. The method of claim 39 or40, wherein the microparticle is a nylon microparticle, a goldmicroparticle, or a magnetic microparticle.
 42. The method of any ofclaims 22-41, wherein the reaction buffer comprises Tris-Cl, magnesiumchloride, ammonium sulfate, ATP, and methanol.
 43. The method of any ofclaims 22-42, wherein the reaction buffer comprises about 100 mMTris-Cl, about 10 mM magnesium chloride, about 5 mM ammonium sulfate,about 1 mM ATP, pH about 7.4, optionally in about 20% v/v methanol. 44.The method of any of claims 22-42, wherein the reaction buffer comprisesabout 50 mM Tris-Cl, about 10 mM magnesium chloride, about 10 mMammonium sulfate, about 4 mM DTT, pH about 7.5, optionally furthercomprising about 0.1 mM to about 1 mM ATP.
 45. The method of any ofclaims 22-44, wherein the test strip comprises filter paper, optionallyfilter paper comprising a pore size of about 11 micrometers.
 46. Themethod of any of claims 22-45, wherein the test strip compriseschitosan.
 47. The method of any of claims 22-46, wherein the pathogen ishuman papillomavirus; the padlock probe comprises a polynucleotidesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:20, and a variant having at least 90% sequenceidentity to any of the foregoing; the primer comprises thepolynucleotide sequence of SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:21, or avariant having at least 90% sequence identity to SEQ ID NO:6, SEQ IDNO:19, or SEQ ID NO:21; and the reporter probe comprises thepolynucleotide sequence of SEQ ID NO:5 or a variant having at least 90%sequence identity to SEQ ID NO:5, or a polynucleotide corresponding to aregion of SEQ ID NO:2 or SEQ ID NO:20.
 48. The method of any of claims22-46, wherein the pathogen is Chlamydia tracomatis; the padlock probecomprises a polynucleotide sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and a variant having at least90% sequence identity to any of the foregoing; the primer comprises thepolynucleotide sequence of SEQ ID NO:12 or a variant having at least 90%sequence identity to SEQ ID NO:12, and the reporter probe comprises thepolynucleotide sequence of SEQ ID NO:11 or a variant having at least 90%sequence identity to SEQ ID NO:11, or a polynucleotide corresponding toa region of SEQ ID NO:8.
 49. The method of any of claims 22-46, whereinthe pathogen is Neisseria gonorrhoeae, the padlock probe comprises apolynucleotide sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and a variant having at least 90%sequence identity to any of the foregoing; the primer comprises thepolynucleotide sequence of SEQ ID NO:18 or a variant having at least 90%sequence identity to SEQ ID NO:18, and the reporter probe comprises thepolynucleotide sequence of SEQ ID NO:17 or a variant having at least 90%sequence identity to SEQ ID NO:17, or a polynucleotide corresponding toa region of SEQ ID NO:14.